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Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m2G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m2G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3–TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3′-CCA of mature tRNAs. We also showed that m2G7 of tRNATrp was introduced by THUMPD3–TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3-knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m2G6/7 modification and pave a way for further studies of the role of m2G in sperm tRNA derived fragments.

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides, which greatly increase the complexity and utility of RNAs. Transfer RNAs (tRNAs) are the most heavily modified cellular RNA molecules and contain the largest variety of modifications. Modification of tRNAs is pivotal for protein synthesis and also precisely regulates the noncanonical functions of tRNAs. Defects in tRNA modifications lead to numerous human diseases. Up to now, more than 100 types of modifications have been found in tRNAs. Intriguingly, some modifications occur widely on all tRNAs, while others only occur on a subgroup of tRNAs or even only a specific tRNA. The modification frequency of each tRNA is approximately 7% to 25%, with 5-20 modification sites present on each tRNA. The occurrence and modulation of tRNA modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered. In particular, tRNA modifications are responsive to environmental changes, indicating their dynamic and highly organized nature. In this review, we summarized the known occurrence order, cross-talk, and cooperativity of tRNA modifications.

The design and fabrication of highly active, robust and cost-efficient electrocatalysts for both oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) is of great significance towards overall water splitting, but remains challenging as well. Herein, we report, for the first time, heterostructural Co/CeO2/Co2P/CoP@NC dodecahedrons as bifunctional electrocatalyst, in which abundant interfaces are formed between different components. Typical ZIF-67 (ZIF = zeolitic imidazolate framework) dodecahedrons with pre-inserted CeO2 nanowires were selected as precursors to synthesize Co/CeO2/Co2P/CoP@NC via a direct carbonization process followed by phosphidation, simultaneously generating the strong coupled heterojunction interfaces through interactions between CeO2 and CoxP species. Abundant porous structure leads to the exposure of more active sites and the carbon encapsulation of nanodomains sustains the high robustness and conductivity and the synergistic effect between the multi-components heterostructure. Benefiting from the above collective advantages, the Co/CeO2/Co2P/CoP@NC electrocatalysts exhibit small overpotentials of 307 and 195 mV to derive 10 mA cm−2 for OER and HER, respectively. Furthermore, an alkaline electrolyzer assembled by using Co/CeO2/Co2P/CoP@NC as both cathode and anode can achieve a current density of 10 mA cm−2 at a low voltage of 1.76 V and work continuously for over 15 h. This work would provide a rational protocol for fabrication multi-phase interface enriched electrocatalysts toward highly efficient energy conversion.

Gelatin methacryloyl (GelMA) has been widely used in bone engineering. It can also be filled into the calvarial defects with irregular shape. However, lack of osteoinductive capacity limits its potential as a candidate repair material for calvarial defects. In this study, we developed an injectable magnesium–zinc alloy containing hydrogel complex (Mg-IHC), in which the alloy was fabricated in an atomization process and had small sphere, regular shape, and good fluidity. Mg-IHC can be injected and plastically shaped. After cross-linking, it contents the elastic modulus similar to GelMA, and has inner holes suitable for nutrient transportation. Furthermore, Mg-IHC showed promising biocompatibility according to our evaluations of its cell adhesion, growth status, and proliferating activity. The results of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining, and real-time polymerase chain reaction (PCR) further indicated that Mg-IHC could significantly promote the osteogenic differentiation of MC3T3-E1 cells and upregulate the genetic expression of collagen I (COL-I), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2). Finally, after applied to a mouse model of critical-sized calvarial defect, Mg-IHC remarkably enhanced bone formation at the defect site. All of these results suggest that Mg-IHC can promote bone regeneration and can be potentially considered as a candidate for calvarial defect repairing.

Urinary crystals in normal and kidney stone patients often have varying sizes; the interaction between renal epithelial cells and COD crystals generated in the tubular fluid could play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. This study aims to compare the cytotoxicity of micro/nano-calcium oxalate dihydrate (COD) crystals (50 nm, 100 nm, 600 nm, 3 μm, and 10 μm) toward African green monkey renal epithelial (Vero) cells to reveal the mechanism of kidney stone formation at the molecular and cellular levels. Methods: Vero cells were exposed to COD crystals of varying sizes at a concentration of 200 μg mL−1 for 6 h. The effects of COD crystals on Vero cell viability, apoptosis rate, and cellular biochemical parameters [lactate dehydrogenase (LDH), superoxide dismutase (SOD), reactive oxygen species (ROS), hyaluronic acid (HA), osteopontin (OPN), and mitochondrial membrane potential (Δψm)] were determined using biochemical and morphological analyses. Results: Vero cell viability and apoptotic rate were closely associated with the size of COD crystals; lower cell viability and higher apoptosis rate were observed in cells exposed to smaller COD crystal size. The expression of SOD, ROS, HA and OPN also changed in a size-dependent manner after exposure to the five different sizes of COD crystals. The area ratio of the (100) face with a high density of Ca2+ ions to the total surface area was also found to influence the severity of cell injury. Cell injury induced by COD crystals was mainly caused by excessive expression of intracellular ROS and reduction of free-radical scavenger SOD. Moreover, binding of large crystals on the cell membrane surface takes more time to cause cell injury than internalized small-sized crystals. The cell death rate was found to be positively correlated with the amount of internalized COD crystals. Conclusions: although the COD toxicity is often disregarded, the size-dependent cytotoxicity of COD crystals toward Vero cells is demonstrated in this study.

The solubilities of 2-naphthalenesulfonic acid monohydrate and sodium 2-naphthalenesulfonate in sulfuric acid solutions were measured at temperatures ranging from 278.15 to 338.15 K by using a dynamic method. The concentration of sulfuric acid solution ranged from 0 to 80wt%,. The solubilities of 2-naphthalenesulfonic acid monohydrate and sodium 2-naphthalenesulfonate increased with temperature, and both of them were the lowest at 70wt%, of sulfuric acid solution(w 3 0 =0.70)while the highest in pure water. The solubility data were correlated by the modified Apelblat equation. Based on the solubility difference between 2-naphthalenesulfonic acid monohydrate and sodium 2-naphthalenesulfonate, a new technique in which sodium sulfate was used to replace sodium sulfite in the neutralization reaction was developed. The suitable mole ratio of H2O to Na2SO4 in the neutralization reaction was 80:1, and that of 2-naphthalenesulfonic acid monohydrate to Na2SO4 was 3.2:1. The material balance under the suitable mole ratios was given and discussed.

To separate a salt mixture (mainly consisting of Na2CO3 and Na2SO4) which is formed from the 4,4′-diaminostilbene-2,2′-disulfonic acid wastewater treatment, the solubility data of Na2CO3 and NaHCO3 in Na2SO4–water solvent mixtures were measured using a dynamic method over the 275–335 K temperature range. The solute-free mass fraction of Na2SO4 in the solvent mixtures extended from 0 to 0.25. The solubility of Na2CO3 and NaHCO3 in Na2SO4–water solvent mixtures decreased with addition of Na2SO4. With rising temperature, the solubility of NaHCO3 increased while the solubility of Na2CO3 increased at first and then decreased after transition points. The experimental data were correlated with the electrolyte nonrandom two-liquid model. The root-mean-square deviations of solubility temperature varied from 0.35 to 1.50 K. Based on the solubility data of this work, a new strategy for separating the Na2CO3 and Na2SO4 mixture was proposed theoretically.

The solubility of 4-chloro-2,5-dimethoxynitrobenzene (CDMB) and 4-chloro-2,5-dimethoxyaniline (CDMA) in methanol, ethanol, xylene and toluene was measured over the temperatures range from (278 to 338) K by the dynamic method using a laser monitoring observation technique. The solubility in all solvents increased with temperature and the greatest solubility of both systems was obtained in toluene. The Wilson and the NRTL models were applied to correlate the experimental results. The root-mean-square deviations for the system of (CDMB + solvent) ranged from T = (0.11 to 0.34) K and (0.08 to 0.33) K calculated by the Wilson and the NRTL models, respectively, while for the system of (CDMA + solvent) the root-mean-square deviations ranged from T = (0.11 to 0.32) K and (0.14 to 0.33) K. The melting points and enthalpies of fusion of CDMA and CDMB were determined by differential scanning calorimetry (DSC). Toluene was found to be the preferred solvent for the reduction of CDMB to CDMA from the point of view of reaction and product separation

The chemical composition of urinary crystallites of less than 1000 nm from 10 calcium phosphate (CaP) stone-formers were investigated and compared with that from healthy subjects using X-ray power diffraction, Fourier transform infrared spectroscopy and nanoparticle size analyzer. Although there were some calcium oxalate monohydrate (COM) crystals in CaP stones, the main components of crystallites in urine of CaP stone formers were uric acid (UA), CaP and COM, while that in healthy urine was mainly UA and a small amount of COM. That is, the CaP content in urinary crystallites of CaP stone-formers was significantly higher than that of controls. The particle size range of crystallites in lithogenic urine was 3~1000 nm and most of these crystallites were aggregated, but it was 30~500 nm in healthy subjects.

Calcium oxalate dihydrate (COD) nanoparticles with an average size of about 200 nm was prepared by complex precipitation method using potassium oxalate (K2Oxa) and CaCl2 as raw materials, NTA as complexing agent. The effects of initial reactant concentrations, pH in reaction system and dispersion media on morphology and size of the nanoparticles were investigated. The samples were characterized by X-ray diffraction (XRD), Fourier transform infrared resonance (FTIR), zetasizer Nano-ZS and transmission electron microscopy (TEM). The results indicated that the prepared sample was COD and its morphology was nearly all elliptical.

This paper investigated the inhibitory effect of degraded soybean polysaccharide (DPS) on the growth of calcium oxalate (CaOxa) crystals. The results were compared with that of soybean polysaccharide without degradation (SPS). The data showed that DPS exhibited a much higher efficiency to inhibit CaOxa growth and stabilize calcium oxalate dihydrate (COD) compared with SPS. As DPS concentration increased, the soluble Ca(2+) ions significantly increased, the aggregation degree of calcium oxalate monohydrate (COM) crystals decreased, the shape of COD crystals became round and blunt, and the Zeta potential on CaOxa crystal surface reduced. The above results were all conducive for the inhibition of CaOxa crystallization. In addition, DPS displayed a distinct repairing effect on oxidative injured renal epithelial cells in African green monkey (Vero), with enhanced cell viability and extracellular superoxide dismutase activity after repair. The morphologies of the repaired cells and their regulatory capability on CaOxa growth were between the control and injured cells. The results indicated that the risk of stone formation can be reduced by DPS, and that DPS may be a potential green drug to prevent the formation of CaOxa stones.

The binding and subsequent fate of 125I-labeled bombesin was studied in rat pancreatic acini. At both 37 degrees C and 4 degrees C binding occurred to a single saturable site, with Kd equal to 1.7 nM. The amount of steady-state tracer binding was reduced at 4 degrees C (5.7%/mg protein) compared with 37 degrees C (8.7%/mg), with a similar relative change in calculated binding capacity. With the use of an acid wash procedure to remove surface bound ligand, 55% of cell-associated 125I-bombesin was internalized in the steady state at 37 degrees C but only 5% at 4 degrees C. Preincubation at 4 degrees C followed by an increase to 37 degrees C led to rapid internalization of bound bombesin, which was blocked by the metabolic inhibitor antimycin. 125I-bombesin was found to be degraded by two acinar systems. One was not related to receptor binding but could be inhibited with bacitracin. The other occurred after internalization and was partially blocked with chloroquine. Thus, after binding, bombesin is internalized, and the degradation products are released from the cell. Exposure to bombesin is also accompanied by a subsequent decrease in cell surface binding (54% after 1 h exposure to 100 nM bombesin), suggesting that the bombesin receptor may also be internalized in a ligand-dependent manner.

The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5–IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1–100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1–10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1–100 nM) induced an early peak of 1,4,5–IP3 at 5–15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5–IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects.

1. The distributions of gastrin- and cholecystokinin-like immunoreactivities in the dog and cat vagus nerves have been studied after nerve section and ligation. 2. In dogs, there was an increase in cholecystokinin-octapeptide-like immunoreactive material on the cranial side of ligatures on the thoracic or cervical vagi. When pairs of ligatures were tied on the cervical vagi there was accumulation proximal, and a slight decrease distal to, the upper ligature. There was also a modest increase distal to the lower ligature. 3. In cats, section of the vagus above the nodose ganglion, and hence degeneration of the efferent fibres, did not prevent increases in cholecystokinin-octapeptide-like immunoreactivity on the cranial side of ligatures which were later tied below the ganglion. Removal of the superior cervical ganglion had no effect on the accumulation of immunoreactive material above the ligatures. Section of the vagus below the nodose ganglion, and hence degeneration of both afferent and efferent fibres, abolished the accumulation on the cranial side of ligatures which were later tied below the section. Cholecystokinin-octapeptide-like material is therefore localized to afferent fibres with cell bodies in the nodose ganglion. 4. Immunoreactive forms were characterized by gel filtration and ion exchange chromatography, and the use of region-specific antisera. In all cats, and all but one dog, a molecule with the properties of sulphated cholecystokinin octapeptide was found to predominate. In some cats (30%) and dogs (26%) a molecule with the properties of heptadecapeptide gastrin (G17) was identified; concentrations of G17 were generally low compared with cholecystokinin octapeptide. In three dogs (20%) there was an accumulation of heptadecapeptide gastrin above the ligatures. 5. Axonal transport of cholecystokinin octapeptide in the vagus is consistent with a neuro-regulatory role for this peptide. However, the functional significance of its localization in afferent fibres, and transport towards the periphery, remains to be determined.

The relationships between molecular forms of gastrin in dog antral mucosa and those in plasma were examined by radioimmunoassay using two regionspecific antisera, and by gel filtration. One antiserum reacts only with intact G-17, the other is Cterminal specific and reacts with G-17, G-34 and their C-terminal fragments. Antral extracts contained predominantly G-17-like immunoreactivity. However, when release of gastrin in conscious or anesthetized dogs was stimulated by feeding, electrical vagal stimulation, instillation of liver extract into the stomach, or by cholinergic drugs

Publisher Summary This chapter discusses the role of cholecystokinin octapeptide in the gut. Extracts of the whole thickness of gut wall contain material derived from both mucosal endocrine cells and enteric neurons. To study neuronal CCK free of contamination by endocrine cell material, the advantage of the fact that the longitudinal muscle of guinea pig ileum and jejunum can be stripped from the underlying circular muscle either with or without the adherent myenteric plexus is gained. In guinea-pig jejunum concentrations of immunoreactive CCK in innervated longitudinal muscle were 34 ± 6 pmol/g compared with 138 ± 37 pmol/g in mucosa. There was a sharp distal gradient in mucosal concentrations but a less markedly gradient in the myenteric plexus, so that in ileum, the concentrations in the longitudinal muscle plus plexus were 14 ± 2 pmol/g compared with 5 ± 1 pmol/g in mucosa. Immunoreactive CCK was localized to the myenteric plexus, rather than the muscle itself, as in denervated strips of muscle, the concentrations were reduced by over 70%. The concentrations of CCK-like peptides in the myenteric plexus are relatively low compared with other neuropeptides, substance P, and vasoactive intestinal polypeptide-like immunoreactivity.